Summary Methylation is a type of epigenetic change: a non-sequence modification of DNA linked to overall changes in gene expression. Global DNA hypomethylation is a common aberrant feature of breast cancers, and is generally thought to contribute to cancer by activating oncogenes, promoting genomic instability and inducing aneuploidy. It is suspected that healthy women with aberrant DNA methylation in normal-appearing breast tissue are at increased risk of breast cancer, and that somatic methylation defects arising in multiple tissues may indicate increased susceptibility to cancer. Studying DNA methylation directly in breast tissue of women is not feasible in epidemiologic studies. Leukocytes are readily accessible, and there is accumulating evidence that peripheral white blood cell (WBC) DNA contains epigenetic information that can be exploited to provide information on an individual's risk of cancer, including breast cancer. In a preliminary case-control study, we have observed a threefold increase in breast cancer risk among women with global hypomethylation in leukocytes as measured by the 5-methyldeoxyctyosine (5-mdC) liquid chromatography-mass spectrometry assay, independent of other breast cancer risk factors. The main goal of this prospective study is to examine the extent to which global DNA hypomethylation, as measured by total 5-mdC content in leukocytes, is related to subsequent breast cancer. We propose to conduct this work in a random subset of 430 postmenopausal breast cancer cases and a total of 430 frequency-matched controls from the Etiology and Early Markers (EEMS) established study of breast cancer nested in the NCI Prostate Lung Colorectal Ovary (PLCO) Screening Trial cohort. Because this is a prospective study with DNA specimens collected up to twelve years before diagnosis, the findings from this study will provide important information on the extent to which epigenetic marks predict an individual's risk of developing breast cancer. This contribution has significant potential to:1) identify women at increased risk o breast cancer who can benefit from additional screening and other prevention strategies; 2) stimulate the search for additional epigenetic risk markers; and 3) improve the characterization of the methylome of high-risk individuals to guide future research into causal mechanisms leading to new prevention strategies.